86 H. HALVORSON et Cll. 



recognized by extrapolating the metabolism of alanine to time 

 zero. The collective literature on spores suggests that a specific 

 receptor site exists on the spore which, following combination 

 with L-alanine, leads eventually to germination. This view led us 

 over 6 years ago to start a search for the identity of the L-alanine 

 binding site on spores of B. cereus. The discovery of an active 

 alanine racemase^^ suggested this as the active site. 



This was ruled out, however, since not only is L-alanine 

 dependency on germination of various strains independent of 

 alanine racemase activity, but also spore germination proceeds 

 under conditions in which the enzyme is inactive^^. 



The amount of L-alanine required to initiate germination is 

 small. When spores are exposed for 45 seconds to ^^C-L-alanine 

 the binding of 200 molecules of L-alanine is sufficient to enable 

 40% germination^-^. Later it was observed-^' ^^ that NH3 and 

 pyruvate release followed L-alanine disappearance. The rate of 

 NH3 release from L-alanine is dependent on heat activation 

 while the amount of NH3 and pyruvate formed, when pyruvate 

 oxidation is blocked by arsenite, is greater than that expected 

 from the L-alanine added^^. 



In order to estimate the endogenous contribution to the 

 release of NH3, the recovery of labeled NH3 from ^-^N-L-alanine 

 was foUowed^^. After 10 min incubation with spores, over 90% 

 of the NH3 was derived from endogenous sources. Employing 

 i^C-labeled alanine and an arsenite block for pyruvate, 89 % of 

 the pyruvate recovered was derived from endogenous sources. 

 The equimolar pyruvate and NH3 recoveries suggested that 

 compounds identical to or closely related to alanine are released. 

 This was further confirmed by reisolation of the alanine from 

 the medium in the absence of arsenite and demonstrating a 50% 

 decrease in the specific activity of the exogenous alanine. Such 

 dilutions are not unexpected since from the work of Powell and 

 Strange"^^, activation and germination are accompanied by a 

 depolymerization of the outer layers of spores which are rich in 

 alanine. 



From the isotope studies the conversion of L-alanine to 



